In recent years, fluorescence image-guided surgery (FIGS) with contrast provided by 5-aminolevulinic acid (5- ALA)-induced protoporphyrin IX (PpIX) has been demonstrated to improve surgical outcomes for glioma patients. However, there are limitations to this wide-field (low-resolution) imaging method. Specifically, it is diffiult to accurately identify a surgical margin based on subtle variations of fluorescence intensity in gliomas, which are diffuse and lack a distinct transition from tumor to normal tissue. Furthermore, wide-field (low- resolution) approaches, such as FIGS and MRI, provide pixel intensities that represent an average value from many cells, resulting in a diminished ability to detect the sparse tumor cell populations at the glioma periphery. This problem is exacerbated in low-grade gliomas, where 5-ALA-induced PpIX fluorescence is only generated by rare proliferating cell populations and is typically undetectable via wide-field FIGS. Recently, Dr. Nader Sanai, a collaborator on this project, has shown that intraoperative cellular-resolution confocal microscopy can be used to visualize these sparse fluorescent cells in low-grade glioma patients treated with 5-ALA. The implications of this finding are highly significant since reported rates of gross-total resection (GTR) have been suboptimal for low-grade gliomas (14% to 46%), suggesting a need for improved image-guidance techniques. Here, we propose to develop a hand-held intraoperative confocal microscope to visualize 5-ALA-induced PpIX expression in low-grade glioma tissues. Ex vivo imaging studies with clinical specimens will demonstrate that this real-time in vivo imaging technique 1) quantifies PpIX expression accurately compared to gold-standard histopathology, 2) has the sensitivity to identify tumor infiltration beyond conventional radiographic margins based on T2-weighted MRI, and 3) provides superior image quality compared to the only existing optical- sectioning microscope in neurosurgical use, the Zeiss Optiscan(R) confocal microscope. Finally, a first-in-human study is proposed to demonstrate the feasibility of incorporating our device into the operative workflow, providing a real-time quantitative optical biopsy that complements existing wide-field imaging techniques in neurosurgery and helps to calibrate surgical decision-making at the final stages of tumor resection. Preclinical and clinical studies will leverage the existing expertise of Dr. Nader Sanai at the Barrow Neurological Institute (BNI), which is the highest-volume operative center for glioma resection in the United States and the only institution with extensive experience using both wide-field FIGS and intraoperative confocal microscopy. The results of these technological developments and translational studies will justify future clinical trials investigating quantitatie intraoperative confocal microscopy as a surgical strategy to maximize extent of resection, minimize operative morbidity, and improve overall survival for low-grade glioma patients.